Genome-wide Collection of Lenti 3’UTR Reporters for Functional Validation of microRNA Targets

Sigma® Life Science and SwitchGear Genomics™

Introduction

microRNAs (miRNAs) are a class of naturally occurring small non-coding RNA molecules that regulate a variety of developmental and physiological processes. To better understand miRNA-UTR interactions, Sigma Life Science has partnered with SwitchGear Genomics to create a genome-wide collection of 10,000 3’ UTR lenti-based luciferase reporters. This collection can be used to validate miRNA targets identified by prediction algorithms or other experimental methodologies.

Here we present data on a screen of a large set of predicted miR-122 targets using 3’ UTR luciferase GoClone™ reporters and validation of several known mRNA targets of human miRNAs using the MISSION® 3’UTR Lenti Goclones and microRNA mimics.

log2-ratio-of-targeting-non-targeting-mirna

Figure 1.SwitchGear Genomics has made an extensively validated set of 3’UTR plasmid based constructs. This genome-wide set has been a proven tool for screening and validation of many different gene targets. Above is data from a screen of a large set of predicted miR-122 targets using 3’ UTR luciferase GoClone reporters and identified several novel targets. Recently, this same 3’UTR content has been used to create a genome-wide library in lentivirus format

Repression of Lenti GoClone™ Activity Using miRNA Mimics with Published Human miRNA Targets

microrna-mimics-knockdown-reporter-activity

Figure 2.microRNA Mimics Knockdown Reporter Activity of Stable MCF7 Cells Transduced with Lenti GoClones. Stable MCF7 cells transduced with Lenti GoClones were transfected with a final concentration of 50nM MISSION® Human microRNA Mimics, in parentheses (red bars) and with MISSION miRNA Negative Control 1 (white bars). Twenty-four hours post-transfection cells were assayed for Renilla luciferase activity using the LightSwitch™ Assay System on a SpectraMax® L Microplate Reader. This was done with four replicates, and the values were plotted as a percent expression compared to GoClone plus negative control as 100 percent expression

Stable Cell Line Creation with MISSION 3’UTR Lenti GoClone Workflow —a Renewable Resource for miRNA Validation

renilla-luciferase-reporter-activity

Figure 3.Renilla Luciferase Reporter Activity Detected in Stable MCF7 Cells Transduced with Lenti GoClones. Puromycin-resistant MCF7 cells were assayed for Renilla luciferase activity using the LightSwitch™ Assay System on a SpectraMax® L Microplate Reader. This was done with four replicates and luciferase output plotted as a value of relative luciferase units (RLU)

MISSION 3’UTR Lenti GoClones Respond Specifically to hsa-miR-122 Validated Gene Targets

mission-3utr-lenti-goclones

Figure 4.MISSION 3’UTR Lenti GoClones were assessed for specificity and performance by analyzing previously identified and validated targets for hsa-miR-122. HT1080 cells were transfected with Lenti GoClones for RIMS1, GNDPA2, LUZP1, or non-targets for hsa-miR-122, which included two random 3’UTR sequences, GAPDH, and vector containing no insert. 24 hours after transfection, cells were treated with 50nM MISSION miRNA mimic for hsa-miR-122 or MISSION miRNA Negative Control 1. Knockdown of the target was measured by taking the log2 of miR-122 mimic/non-targeting control

Conclusions

  • Validated miRNA target knockdown in both plasmid and lentiviral-based UTR reporter constructs
  • MISSION 3’ Lenti GoClones allow integration into cell lines recalcitrant to transfection and the creation of stable 3’UTR reporter cell lines
  • Genome-wide pre-cloned 3’UTR sequences provide researchers measurable savings in both time and money
Materials
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©2012 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. MISSION is a registered trademark of Sigma-Aldrich Co. LLC. SwitchGear Genomics, LightSwitch and GoClone are trademarks of SwitchGear Genomics. SpectraMax is a registered trademark of Molecular Devices Corp.

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